Commercial forestry is concerned almost exclusively with conifers, especially trees of the Spruce genus (e.g. the Norway Spruce Picea abies), the Pine genus (e.g. the Loblolly pine, Pinus taeda), the Douglas Fir (Pseudotsuga menziesii) and the Larch (Larix sp.).
Their commercial significance has prompted much research into the cultivation and growth of such conifers. This research has been aided by the development of in vitro tissue culture systems, allowing for the cloning of selected strains.
At present, there are two main methods of producing multiple clones of coniferous trees.
One method, organogenesis, involves taking a suitable explant and cultivating the tissue in culture media containing carefully controlled nutrient and hormone levels, resulting in the formation of callus. This callus can then be placed on budding media. The resulting buds are separated and can be grown to form individual plantlets (as described by von Arnold, 1984).
The other method routinely employed is that of somatic embryogenesis, as disclosed by von Arnold & Hackman (1988) and by WO 91/05854. This method, less labour intensive than organogenesis, involves the formation in vitro of embryogenic callus from zygotic embryo explants. This callus gives rise to many proembryonic structures which can be separated and grown into plantlets.
However, somatic embryogenesis can only be performed using embryos or young cotyledon as starting materials. For example, success has been achieved with cotyledon explants from seven day old Picea abies seedlings (Lelu et al., 1984) and 30 day old seedlings of Picea mariana and Picea galuca (Attree et al., 1990) but not from older trees.
Similarly, organogenesis is fully successful only when the explant is taken from very young seedlings. When explants from older plants are used, it is possible to obtain adventitious buds, but these cannot be grown into plantlets (Jansson & Bornman, 1983; von Arnold, 1984).
This represents a problem because it is impossible to predict the quality of the mature tree from a young seedling or plantlet. Thus one might be cloning an inferior specimen. The present invention aims to overcome this problem.